One of the major challenges facing the drug discovery process is the identification of small organic ligands that will bind to target species, particularly protein targets. A multitude of new protein targets are being discovered by genomics and bioinformatics efforts. Many of these proteins have no known function or known specific ligands. Thus, the identification of ligands for these targets presents challenges in the screening of large chemical libraries by high throughput screening (HTS), including ultra-high throughput screening (UHTS), methods, particularly from the standpoint of assay development. Hence, there is a need for a straightforward, generally applicable methodology, particularly an HTS assay methodology, that can be used to identify ligands that bind proteins, especially those with unknown functionality.
It is known that the binding of substrates or specific ligands does, in general, alter the intrinsic stability and hence the denaturation profile of a protein. Thus, methods that measure protein denaturation can be used to detect and quantitate ligand-protein interactions.
The denaturation of proteins is accompanied by the progressive loss of their tertiary/quartenary structure and ultimately biological activity. Denaturation can be accomplished by a number of physical and chemical methods that involve changes in temperature, pH, and/or ionic strength, use of chaotropic agents, etc. It can be followed by methods sufficiently sensitive to monitor conformational changes in a protein. Because it is a simple and widely applicable experimental method, thermal denaturation has been used for a variety of purposes, including purifying proteins by selective denaturation of impurities and to study protein structure, folding, and stability. Thermal denaturation curves ((TDC), where the fraction of denatured protein is measured as a function of gradually increasing temperatures) obtained by differential scanning calorimetry (DSC) have been shown to be particularly useful for determining protein stability and making inferences about the tertiary structure. The usefulness of TDC is further enhanced because binding of compounds that are substrates or specific ligands for a given protein changes the intrinsic stability of that protein and, hence, causes a shift in the TDC and the Tm (midpoint temperature) values.
Interpretation of the results of thermal scanning methods depends on the assumption that the denaturation process is a one-step, reversible, and continuous process that is very rapid on the time scale of the temperature scanning rate. However, the denaturation of most proteins under the usual experimental conditions is irreversible. Typically, it is only with small proteins and very mild denaturing agents that denaturation is readily reversible. Thus, DSC may be unable to provide reproducible and readily interpretable binding measurements.
In general, the DSC curves reflect the stability of many different structural domains, some sensitive to the binding of ligands and some not sensitive at all. Furthermore, denaturation may be initiated at many locations within the protein structure. Each of these processes has its own activation energy, which makes it the dominant process only within a narrow temperature range. As a consequence, depending on the scanning rate, the stability of a given domain may or may not be evident in the DSC curve. Furthermore, differential scanning calorimetry may see two or more protein denaturation steps where one would expect only a single transition. Yet another major factor contributing to the greater inextricability of the scanning thermal denaturation methods is that the binding equilibria of both the ligands of interest and of the fluorescent dyes reporting on the structural integrity of the protein are strongly temperature dependent. Thus, both the sensitivity of the method and the stabilizing effect of the ligand under study drift drastically during the experiment.
Therefore, a need exists for a method identifying compounds that bind to target species. Preferably, such a method is amenable to UHTS or HTS, reproducible, and independent of the heating rate.
The present invention provides methods for identifying compounds that bind to target species (e.g., polypeptides including proteins, and polynucleotides including DNA and RNA). These methods involve the use of isothermal denaturation, preferably in combination with fluoresence detection methods. Significantly, the methods of the present invention involve automated methods suitable for HTS and UHTS. Ideally, the methods of the present invention arc envisioned to be scalable to evaluate 10,000-60,000 compounds or more in a 24 hour period.
Isothermal denaturation of proteins offers an attractive method for the identification of binding ligands. Significantly, in preferred methods, the present invention couples fluorescence techniques with denaturation by isothermal methods to determine alteration of target (e.g., protein) stability by a bound ligand. In particularly preferred embodiments, the denaturation and stabilization or destabilization of target species (e.g., protein targets) by ligands against isothermal denaturation is quantified by changes in fluorescence intensity.
In one preferred embodiment, the present invention provides a method for identifying a test compound that binds to a target species. The method includes: incubating at least one test mixture (preferably, a plurality of test mixtures for high throughput screening) under isothermal denaturing conditions, each test mixture comprising at least one test compound (preferably, at least two test compounds and more preferably, twp to ten test compounds), and at least one target species (preferably, only one target species is in any one test mixture), wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature (e.g., unfold) to a measurable extent; detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions. Typically and preferably, the methods of the present invention can evaluate at least about 100 test mixtures per day. Preferably, such an evaluation occurs substantially simultaneously.
In the methods described herein, the target species can be a polypeptide (e.g., protein) or a polynucleotide (e.g., DNA or RNA). Preferably, the target species is a protein. The compound can bind to the target species either specifically (e.g., at a specific site or in a specific manner) or unspecifically. The binding can involve a variety of mechanisms, including covalent bonding, ionic bonding, hydrogen bonding, hydrophobic bonding (involving van der Waals forces), for example, or combinations thereof.
In the present invention the following definitions apply:
Isothermal denaturing conditions refers to conditions effective to denature a target molecule at a fixed temperature. It can also involve defined conditions with respect to pH, ionic strength, cation concentration, etc., which are generally held constant for evaluation of various compounds for a given target.
Denaturation signal refers to the signal produced by the target species upon being denatured.
Tm refers to the midpoint of the melting transition of the target as determined by differential scanning calorimetry.
Reporter molecule refers to a separately added molecule such as a fluorescent dye or a covalently bonded reporter group attached to the target.